CN103031336A - Cattle plasmid adenovirus vector pAd-ZBED6 as well as construction method and use of cattle plasmid adenovirus vector pAd-ZBED6 - Google Patents
Cattle plasmid adenovirus vector pAd-ZBED6 as well as construction method and use of cattle plasmid adenovirus vector pAd-ZBED6 Download PDFInfo
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Abstract
The invention relates to a cattle plasmid adenovirus vector pAd-ZBED6 as well as a construction method and use of the cattle plasmid adenovirus vector pAd-ZBED6, which belongs to the technical field of gene engineering. The construction method of the cattle plasmid adenovirus vector pAd-ZBED6 comprises the follow steps: (1) inserting Qinchuan cattle ZBED6 gene into a CMV (cytomegalovirus) promoter of a pAdTrack-CMV adenovirus shuttle plasmid to construct a recombinant shuttle plasmid pAdTrack-CMV-ZBED6; (2) after linearization of Pme I, converting an E.coil BJ5183 competent cell containing a pAdEasy-1 plasmid, completing homologous recombination of pAdTrack-CMV-ZBED6 and pAdEasy-1 in bacteria by means of an efficient homologous recombination system of Cre recombinase in the E.coil BJ5183, and naming correct recombinant adenovirus plasmid as pAd-ZBED6; and (3) after Pac I digestion linearization of pAd-ZBED6, transfecting a 293A cell and an original muscle cell of Qinchuan cattle by lipidosomes to obtain recombinant virus particles. The cattle plasmid adenovirus vector pAd-ZBED6 can be applied to functional study and authentication of Qinchuan cattle ZBED6 gene as well as modification of seed cells so as to regulate metabolism of genes related to growth and development of muscle.
Description
Technical field
The present invention relates to a kind of plasmid-type adenovirus carrier of the ZBED6 of containing gene, the structure of this plasmid-type adenovirus carrier adenovirus carrier and in the application of transforming seed cell and ox ZBED6 Function Identification.
Background technology
The AdEasyTM system is a quick system that is used for replacing traditional adenovirus recombination system that is made up by (1998) such as T.C.He.In this system, only needed for two steps can produce recombinant adenovirus: the transfer vector of first expression cassette being packed into, and then by homologous recombination insertion adenoviral gene group.The effective way of foreign gene being inserted adenovirus is by homologous recombination, and reason is: 1) adenovirus DNA is large-scale linear molecule, contains nearly all inscribe restriction enzyme site; 2) genome excessive (36kb) is difficult to operation.
In the AdEasyTM carrier system, the carrier that contains most of adenoviral gene group is super spirial plasmid, rather than linear DNA, and homologous recombination is then carried out intestinal bacteria.For legacy system, these 2 improvement make the viral DNA operation easier, simultaneously owing to having utilized colibacillary high-level efficiency homologous recombination characteristic to make the screening of recombinant vectors more simple.In native system, at first gene cDNA is inserted a transfer vector, with the plasmid that obtains PmeI linearizing, then in E.coli BJ5183, carry out homologous recombination with viral DNA plasmid pAdEasy-1.PAdEasy-1 has lacked E1 and E3 district, and its E1 district function will obtain complementation in the 293A cell.Recon screens by kantlex, carries out identification and analysis with restriction endonuclease.With the recombinant adenovirus that obtains PacI linearizing, expose its inverted terminal repeat (ITR, IavertedTermined Repeats) at last, produce recombinant virus particle behind the transfection 293A cell.
Homologous recombination is carried out between linearizing transfer vector and complete superhelix adenoviral plasmid.Use complete adenoviral plasmid and carry out linearizing without restriction endonuclease, recombinant adenovirus is most important for producing.Kalamycin resistance gene in the transfer vector then can be used to screen recon.Because it is lower that linearizing transfer vector produces kalamycin resistance clone's background, so there is higher signal to noise ratio in this homologous recombination system.E.coli BJ5183 has recA active, but other enzyme of simultaneously disappearance mediation bacterium restructuring has efficient conversion and restructuring ability.In case recon, then can change common bacterial strain without recA, endA activity such as DH5 α etc. over to through determining and increase.Because it is active to lack recA, DH5 α can not be used for the homologous recombination of adenovirus.
Compare with legacy system, screening and required time of purification of adenoviral have shortened greatly in the AdEasyTM system.Clone and screening approximately needed for 1～3 week, needed checking recombinant virus plasmid whether to contain goal gene after the restructuring.Recon changes mammalian cell 293A over to after increasing in DH5 α, at last the recombinant virus particle that produces in the 293A cell is carried out purifying and titration.The recombinant adenovirus that finally obtains can only be bred in the 293A cell that E1 district function is provided.
In general, producing recombinant adenovirus with traditional method does not need extensive work, only needs every day carry out passage and observe plaque test in 1 hour.But because virus plaque formation speed is slow, whole process just needs for a long time.And the AdEasyTM carrier system produces recombinant virus with intestinal bacteria, has greatly shortened required time.
Zinc finger protein is the transcription factor that a class has the finger-shaped structural domain, and gene regulating is played an important role.According to the difference of its conserved domain, zinc finger protein mainly can be divided into C2H: type, C.Type and C6 type.Zinc refers to by being combined with the sequence-specific of target molecule DNA, RNA, DNA-RNA, and with the combination of self or other zinc finger proteins, transcribe with translation skill on the expression of regulatory gene.
Zinc finger protein ZBED gene family has been found 5 members altogether; Its function is as follows: the ZBED1 gene: participate in regulating some and can promote the gene of the propagation of cell; ZBED2 gene: participate in fetal development; ZBED3 gene: participate in just regulating and control Wnt/ β-catenin signal path.ZBED4 gene: act on amphiblestroid formation.ZBED5 gene: with breast cancer related.The ZBED6 gene: major effect growth, cell proliferation and muscle growth.
The ZBED6 gene is the transposon that loses the swivel base function, is arranged in ZC3H11A gene First Intron in all Mammalss; This gene is guarded at mouse, rat, people, orangutan, dog, horse, family's pig camber.The Real-time PCR Analysis demonstration, the mRNA of mouse ZBED6 has to some extent expression at brain, heart, kidney, liver, lung, muscle, ovary, spleen, tail and testis tissue.
ZBED6 albumen contains 2 BED structural domains and 1 hATC dimeric structure territory; The 61st ~ 80 and the 231st ~ 248 amino acids residue are 2 nucleus signal for locatings (rich Lys-Arg sections).This albumen is conservative at the Mammals camber, and especially DNA (comprises a pig) and demonstrates 100% similarity in conjunction with the BED structural domain in 26 species.
Markljung etc. (2009) show with the experimental result of siRNA after with the ZBED6 gene silencing in the mouse C2C12 sarcoplast, ZBED6 albumen is by (insuLin-like growth factor 2, IGF2) QTN (Quantitative traitnucleotide) site in gene the 3rd intron is in conjunction with the expression that suppresses the IGF2 gene with being positioned at IMA-IGF2BP3-001 as supressor.Behind the ZBED6 gene silencing, the expression amount of IGF2 gene raises, cell proliferation (myotube formation) is accelerated, wound healing is accelerated.
Markljung etc. (2009) adopt the ChIP-Seq technology that mouse C2C12 sarcoplast is analyzed, research ZBED6 albumen action target spot (IGF2 gene and downstream gene thereof), the QTN site in the IGF2 gene intron is the main binding site of ZBED6 albumen on genome.The replacement of the pig IGF2 of family gene the 3rd intron list bases G → A, cause the forfeiture of supressor ZBED6 binding site on this gene, thereby make IGF2 up-regulated expression (expression amount is 3 times of normal circumstances) in skeletal muscle, this sudden change major effect muscle growth (improved the quantity of skeletal muscle, thus the pork output increased 3% ~ 4%), cardiac enlargement and fatty deposits.GO note analysis discovery, 1200 action target spots of ZBED6 albumen play the effects such as growth, transcriptional control, cytodifferentiation.Wherein, 262 target gene encoding transcription factors, 36 contain the Homeobox structural domain, and 26 belong to bHLH (basic helix-loop-helix) family, and 10 belong to FOX family, 8 nuclear receptors, 7 belong to SOX family.
In sum, in Mammals, ZBED6 is an important transcription factor, affects growth, cell proliferation and muscle growth.Structure by Qinchuan Cattle ZBED6 gene adenovirus carrier can provide important research data for the breeding of the marker assisted selection of Qinchuan Cattle meat performance, growth performance and new improved seeds, finally for the research of transgenic animal and Qinchuan Cattle grows and the improvement of beef quality provides theoretical foundation.
Summary of the invention
The object of the invention is to disclose a kind of plasmid-type adenovirus carrier pAd-ZBED6.
Second purpose of the present invention has been to disclose the preparation method of plasmid-type adenovirus carrier pAd-ZBED6.
The 3rd purpose of the present invention has been to disclose the application of plasmid-type adenovirus carrier pAd-ZBED6.
The objective of the invention is to be achieved through the following technical solutions:
A kind of plasmid-type adenovirus carrier pAd-ZBED6, wherein, described plasmid-type adenovirus carrier pAd-ZBED6 contains the Qinchuan Cattle ZBED6 gene of nucleotide sequence shown in the SEQ ID NO.1.
The described plasmid-type adenovirus carrier of technique scheme pAd-ZBED6, wherein, described plasmid-type adenovirus carrier pAd-ZBED6 is finished by the AdEasyTM system constructing.
The described plasmid-type adenovirus carrier of technique scheme pAd-ZBED6, wherein, the ZBED6 gene among the described plasmid-type adenovirus carrier pAd-ZBED6 is connected under the shuttle plasmid CMV promotor.
The described plasmid-type adenovirus carrier of technique scheme pAd-ZBED6 wherein, contains the Kozak sequence before the ZBED6 gene start codon among the described plasmid-type adenovirus carrier pAd-ZBED6; Wherein the Kozak sequence is GCCCACC.
The described plasmid-type adenovirus carrier of technique scheme pAd-ZBED6 wherein, contains 6 His sequence labels behind the ZBED6 gene start codon among the described plasmid-type adenovirus carrier pAd-ZBED6.
Make up the method for the described plasmid-type adenovirus carrier of arbitrary technical scheme pAd-ZBED6 in the technique scheme, described method comprises the steps:
(1), the ZBED6 gene fragment with pcr amplification is connected to the pGM-T carrier, recombinant plasmid pGM-T-ZBED6; With recombinant plasmid pGM-T-ZBED6 and shuttle plasmid pAdTrack-CMV through KpnI and XbaI double digestion, utilize T4DNALigase to carry out even reaction of enzyme ZBED6 gene fragment and pAdTrack-CMV carrier segments, enzyme connects product Transformed E .coli DH5 α competence bacterium, obtains the pAdTrackCMV-ZBED6 recombinant plasmid;
(2), restriction enzyme Pme I linearizing pAdTrack-CMV-ZBED6 recombinant shuttle plasmid, contain pAdEasy-1 plasmid E.coliBJ5183 competence bacterium with the conversion of linearizing pAdTrack-CMV-ZBED6 recombinant shuttle plasmid, utilize the efficient homologous recombination of Cre recombinase system in the E.coli BJ5183, in bacterium, finish the homologous recombination of pAdTrack-CMV-ZBED6 and pAdEasy-1, obtain to carry the recombinant adenovirus plasmid pAd-ZBED6 of ZBED6 gene.
The described method of technique scheme, wherein, described method also comprises after recombinant adenovirus plasmid pAd-ZBED6 usefulness liposome LIPOFECTAMINE 2000 transfections, carry out the virus packing in the 293A cell.
The described plasmid-type adenovirus carrier of arbitrary technical scheme pAd-ZBED6 in the technique scheme is as the application of Qinchuan Cattle ZBED6 Functional identification of genes.
The described plasmid-type adenovirus carrier of arbitrary technical scheme pAd-ZBED6 in the technique scheme is as the application of cell transformation.
The described plasmid-type adenovirus carrier of arbitrary technical scheme pAd-ZBED6 in the technique scheme is as the application of muscle growth developmental regulation.
Concrete:
The present invention is achieved through the following technical solutions:
Behind the pcr amplification ZBED6 and the pAdTrack-CMV shuttle plasmid through KpnI and XbaI double digestion, gel reclaims test kit and reclaims ZBED6 gene fragment and pAdTrack-CMV carrier segments, and the carrier segments of recovery and goal gene fragment T4DNA Ligase carry out enzyme and connect reaction.Enzyme connects product Transformed E .coli DH5 α competent cell, purification kit extracting pAdTrack-CMV-ZBED6 recombinant shuttle plasmid, and KpnI and XbaI double digestion assay certificate obtain correct pAdTrackCMV-ZBED6 recombinant plasmid.
Restriction enzyme PmeI linearizing pAdTrack-CMV-ZBED6 recombinant shuttle plasmid, gel reclaims test kit and reclaims linearizing pAdTrack-CMV-ZBED6 recombinant shuttle plasmid, conversion contains pAdEasy-1 plasmid E.coli BJ5183 competent cell, utilize the efficient homologous recombination of Cre recombinase system in the E.coli BJ5183, pAdTrack-CMV-ZBED6 and pAdEasy-1 homologous recombination obtain to carry the recombinant adenovirus plasmid of ZBED6 gene in bacterium, with the recombinant adenovirus plasmid called after pAd-ZBED6 of homologous recombination success.Test kit extracting homologous recombination plasmid, 0.7% agarose gel electrophoresis is selected the recombinant adenovirus plasmid larger than pAdEasy-l, and Pac I enzyme is cut.Enzyme is cut product and is identified by 0.7% agarose gel electrophoresis, and the agarose gel electrophoresis result is with homologous recombination plasmid multiple clone site collection of illustrative plates is identical in theory.PAd-ZBED6 recombinant adenovirus plasmid Transformed E .coli DH5 α competent cell, a large amount of extracting plasmids are for subsequent use.The pAd-ZBED6 recombinant adenovirus plasmid exposes reverse terminal repeat with the PacI linearizing, adopts liposome transfection packing cell (293A cells) to carry out the virus packing.Fluorescent microscope direct viewing behind the transfection 24h, visible shuttle plasmid with green fluorescent protein (GFP) genetic expression.The recombinant virus supernatant liquor infects 293A cell amplification recombinant adenovirus, the expression of fluorescence microscope green fluorescence protein gene.
With recombinant adenovirus plasmid pAd-ZBED6 infected cattle muscle cell of former generation, utilizing real-time quantitative PCR and Western bloting technology for detection to cross and express the impact of the expression pattern of the muscle cell proliferation differentiation of ZBED6 gene pairs ox and muscle development genes involved, is that recombinant adenovirus plasmid pAd-ZBED6 of the present invention is as the application of Qinchuan Cattle ZBED6 Functional identification of genes aspect.
Recombinant adenovirus plasmid pAd-ZBED6 of the present invention, can be used for the infected cattle muscle cell, the beef fat cell, 293 cells, the C2C12 cell, the clones such as 3T3L cell detect it to the impact of corresponding cell structure and function, are the application that recombinant adenovirus plasmid pAd-ZBED6 of the present invention transforms as cell.
With recombinant adenovirus plasmid pAd-ZBED6 infected mice C2C12 clone, utilizing real-time quantitative PCR and Western bloting technology for detection to cross the differentiation of expression ZBED6 gene pairs muscle cell proliferation and the differential expression situation of muscle development marker gene (MyoD, MyoG, MEF-2D, Pax7, MHC) in muscle cell, is that recombinant adenovirus plasmid pAd-ZBED6 of the present invention is as the application of muscle growth developmental regulation.
The present invention has following beneficial effect:
The present invention has made up the recombinant adenoviral vector that can express Qinchuan Cattle ZBED6 gene, the carrier that the present invention is constructed, behind the Qinchuan Cattle muscle cell of the former culture of transfection, can obtain the high efficient expression of ZBED6mRNA, be ZBED6 Functional identification of genes and engineered cells, regulation and control metabolism and grow and lay a good foundation.
Description of drawings:
1, Fig. 1 is the Qinchuan Cattle ZBED6 gene of pcr amplification;
2, Fig. 2 is that pAdTrack-CMV-ZBED6 recombinant plasmid double digestion is identified;
3, Fig. 3 is that pAd-ZBED6 recombinant adenovirus plasmid PacI enzyme is cut evaluation;
4, Fig. 4 is pAd-ZBED6 plasmid transfection 293A cell 5d;
5, Fig. 5 is pAd-ZBED6 plasmid transfection 293A cell 10d;
6, Fig. 6 is pAd-ZBED6 plasmid transfection Qinchuan Cattle muscle cell 4d of former generation.
Embodiment:
For making technical scheme of the present invention be convenient to understand, the application in preparation treatment Gastric Ulcer Treatment is further described to GUIZIHONG below in conjunction with concrete test example.
The present invention utilizes the vitro enzyme of plasmid to cut and interconnection technique, use the pAdEasy-1 system, linearizing shuttle plasmid is transformed the E.coli BJ5183 competent cell that contains the pAdEasy-1 plasmid carry out homologous recombination, the gained recombinant adenovirus plasmid can filter out by kantlex, and identifies by plasmid size and restriction endonuclease analysis.At last, the recombinant adenovirus plasmid transfection 293A cell packing after the PacI enzyme is cut produces recombinant adenovirus.
Embodiment 1:The structure of pAd-ZBED6 recombinant adenovirus plasmid
1, materials and methods:
1.1, instrument:
Bechtop, biochemical cultivation case, gene-amplificative instrament, the single groove gradient of PTC-200 gene-amplificative instrament, Heraeus high-speed refrigerated centrifuge (Germany), Bio-Rad gel image analyser (USA), CO
2Incubator, HZS-H water bath chader (Harbin), Eppendorf pipettor, DYY-III type voltage stabilization and current stabilization electrophoresis apparatus (Beijing 6 1), DYY-III 31A and DYY-III 28D electrophoresis chamber (Beijing 6 1), ice making instrument, MDF-382E Ultralow Temperature Freezer (SANYO GS), Eppendorf table model high speed centrifuge, Sartorious electronic balance (Germany), conventional refrigerator etc.
1.2, main biochemical reagents and test kit:
Long Taq polysaccharase, PrimeSTAR archaeal dna polymerase, DNA restriction enzyme (XbaI, PacI, PmeI, KpnI and XbaI etc.), collagen protein, trypsinase, DNA Marker, dna ligase, Trizol, reverse transcription test kit, expression vector (pAdTrack-CMV, pAdEasy-1), plasmid extraction kit, dna gel reclaim test kit, foetal calf serum, cell culture medium etc.
1.3, substratum and resistance:
LB substratum: Tryptones, yeast powder, NaCl, agar powder;
Resistance: penbritin, card is received mycin etc.
1.4, general reagent:
Heparin sodium, Tris, EDTA, NaCl, NaOH, dehydrated alcohol, sodium-acetate, sodium laurylsulfonate (SDS), microbiotic, ethidium bromide (EB), tetrabromophenol sulfonphthalein, dimethyl benzene cyanines FF, acetic acid, sucrose, deionized formamide, nitric acid, hydrochloric acid, Silver Nitrate, anhydrous sodium carbonate, Sulfothiorine, formaldehyde, boric acid, agarose, KCl, Na
2HPO
4, KH
2PO
4, the saturated phenol of Tris (pH=8.0), chloroform, primary isoamyl alcohol, glycerine, paraffin oil.
1.5, the design of Qinchuan Cattle ZBED6 gene PCR primer (primer is synthetic by giving birth to worker's biotechnology (Shanghai) limited-liability company):
The ZBED6mRNA sequence (NC 007314.4) of announcing with reference to GenBank designs primer:
Upstream primer: 5 '〉CGGggtaccGCCCACCATGcaccaccaccaccaccacAGTGTATGTACCTTAAGTG TACC＜3 ';
Downstream primer: 5 '〉CCCaagcttTTAAGGTAATATTTCTTT TTCATTGC＜3 ',
KpnI wherein, XbaI is respectively the restriction enzyme site of upstream and downstream.
1.6, the ZBED6 gene fragment of pcr amplification Qinchuan Cattle:
(1), PCR total reaction system is 50 μ L, sees Table 1:
Table 1 PCR reaction system of the present invention
(2), the PCR response procedures, see Table 2:
Table 2 PCR response procedures of the present invention
1.7, carry the structure of the pGM-T-ZBED6 recombinant plasmid of ZBED6 gene:
Owing to using the PCR product of high-fidelity enzyme PrimeSTAR amplification not to be with the A tail, can't carry out the TA clone, so need doing, amplified production adds the A processing, just can link on the pGM-T carrier, reaction system sees Table 3:
The table 3 A reaction system that adds of the present invention
Annotate: reaction conditions is 72 degree (suggestion water-bath), 10 minutes.
With the ZBED6 gene fragment of pcr amplification after adding the A reaction; spend the night and be connected through T4Ligase16 ℃ with the pGM-T carrier, obtain the pGM-T-ZBED6 recombinant plasmid, latter's Transformed E .coli DH5 α competent cell; the choosing colony amplification, KpnI and XbaI double digestion were identified after plasmid reclaimed.
KpnI and XbaI enzyme cutting reaction system 20 μ L see Table 4:
Table 4 KpnI of the present invention and Xba I endonuclease reaction system
Annotate: it is 37 ℃ that enzyme is cut digestion condition, 3～10h.
1.8, carry the structure of the pAd-TrackCMV-ZBED6 recombinant plasmid of ZBED6 gene:
KpnI and XbaI double digestion carry pGM-T-ZBED6 recombinant plasmid and the pAdTrack-CMV plasmid of ZBED6 gene, and 16 ℃ of connections of spending the night of T4Ligase after glue reclaims obtain the pAdTrack-CMV-ZBED6 recombinant plasmid.Latter's Transformed E .coli DH5 α competent cell, the choosing colony amplification, KpnI and XbaI double digestion were identified after plasmid reclaimed.
Linked system 25 μ L see Table 5:
Table 5 ligation system of the present invention
Annotate: condition is 16 ℃, 10～12h.
1.9, carry the structure of the pAd-ZBED6 recombinant adenovirus plasmid of ZBED6 gene:
Restriction enzyme PmeI linearizing pAdTrack-CMV-ZBED6 recombinant shuttle plasmid, gel reclaims test kit and reclaims linearizing pAdTrack-CMV-ZBED6 recombinant shuttle plasmid, conversion includes the E.coli BJ5183 competent cell of pAdEasy-1 plasmid, utilize the efficient homologous recombination of Cre recombinase system in the E.coli BJ5183, in bacterium, finish pAdTrack-CMV-ZBED6 and pAdEasy-1 homologous recombination, obtain the recombinant adenovirus plasmid pAd-ZBED6 that carries the ZBED6 gene of homologous recombination success, test kit extracting homologous recombination plasmid, 0.7% agarose gel electrophoresis, the recombinant adenovirus plasmid that selection is larger than pAdEasy-l, the PacI enzyme is cut.Enzyme is cut product and is identified by 0.7% agarose gel electrophoresis, and the agarose gel electrophoresis result is with homologous recombination plasmid multiple clone site collection of illustrative plates is identical in theory; Concrete steps are as follows:
(1), BJ5183 competent cell that 2 pipes are contained pAdEasy-1 places on ice, 1 pipe adds the linearizing pAdTrack-CMV-ZBED6 of 6 μ LPmeI and flicks mixing, another pipe is done empty map, ice bath 30min;
(2), 42 ℃ of water-baths 90 seconds, place rapidly ice to leave standstill 3min;
(3), every pipe adds the LB substratum 1000 μ L of 37 ℃ of preheatings, 60min are cultivated in 37 ℃ of concussions;
(4), conversion product is spread 3 ~ 5 LB/Kan (50 μ g/mL) culture plate, 37 ℃ of cultivation 24h.Spread respectively 100,300 and 600 μ L, obtain the probability of separable bacterium colony with raising, approximately obtain 40 ~ 100 bacterium colonies;
(5), choose 24 best clones, change in 2mL LB/Kan (the 50 μ g/mL) nutrient solution and cultivate 10 ~ 15h.Because producing non-purpose recon, as early as possible extracting recombinant plasmid;
(6), prepare in a small amount plasmid with traditional alkaline lysis, get half and carry out the size that 0.7% agarose gel electrophoresis detects plasmid.The big or small approximately 40kb of recombinant plasmid is because it is low copy plasmid, so answer corresponding adjustment concrete operations when preparing in a small amount;
(7), the bacterium colony more than 20% contains large plasmid (approximately 40kb), these are candidate recons.Contrast with the bacterium colony of contrast culture plate growth and can estimate that the background of linearizing shuttle plasmid due to being connected again is strong and weak;
(8), the screening of recombinant plasmid and amplification
The candidate recon is carried out restriction analysis, verify its structure.The PacI enzyme is cut the small segment of the rear fragment that usually obtains about 30kb and one 3.0 or 4.5kb, and the size of small segment is different at left arm or replicon because of recombinable site.
Select best positive recombinant and change among the E.coli DH5 α and increase, it is essential that this step keeps the structure of recombinant plasmid during for amplification.Because recombinant plasmid is unsettled in BJ5183, and can obtain at an easy rate a large amount of recombinant plasmids in DH5 α.
1.10, contain the liposome LIPOFECTAMINE 2000 transfection 293A cells (namely in the 293A cell, carry out virus packing) of pAd-ZBED6 recombinant plasmid:
(1), transfection the day before yesterday, be inoculated on 6 well culture plates with suitable cell density; During transfection, cell will reach 90%～95% fusion;
(2), solution 1:240 μ L serum free medium+10 μ L LIPOFECTAMINE 2000 (incubation 5min);
(3), solution 2:X μ L serum free medium+4 μ g plasmids (cumulative volume 250 μ L);
(4), solution 1 is mixed room temperature underlying 20min with solution 2;
(5), meanwhile, with the cell in 6 orifice plates with twice in serum free medium flushing cell after, add the 2mL serum free medium;
(6), the mixed solution with solution 1 and solution 2 dropwise adds in the hand-hole wave and culture plate, gently mixing.At 37 ℃, 5% CO
2Middle insulation 5～6h;
(7), behind the 6h, change the full substratum that contains serum, at 37 ℃, 5% CO
2In 24～72h detect the transfection level.
1.11, the evaluation of carrying the recombinant adenovirus of Qinchuan Cattle ZBED6 gene:
Get recombinant adenovirus supernatant 500 μ L, be added in the 90% 293A cell that merges, 37 ℃, 5% CO
2Middle cultivation.After Microscopic observation to 95%～pathology appears in 100% cell, centrifugal collection supernatant.And with cell multigelation 3 times between-80 ℃ and 37 ℃, with the centrifugal collection supernatant of 3000r/min, the adenovirus supernatant is carried out centrifugal purification.Get 5 μ L virus supernatant and add 10 μ L Proteinase Ks, hatch 1h for 55 ℃, boil again 5min, get 2 μ L after centrifugal to be PCR.
1.12, carry the titer determination of the recombinant adenovirus pAd-ZBED6 of Qinchuan Cattle ZBED6 gene:
(1), cell is prepared: inoculation 100 μ L293A cells in 96 orifice plates, every porocyte number approximately 10
4Individual, cultivate with 2%DMEM;
(2), virus dilution liquid is prepared: with 2%DMEM virus liquid is diluted to 8 higher concentrations (such as 10
-3～10
-10), each concentration repeats 10, and every hole adds viral dilution liquid 100 μ L.Other stays two rows not add virus liquid as negative control.Under 37 ℃, incubator was cultivated 10 days;
(3), observation of cell behind the 10d, the hole count of the existing CPE of every discharge that counts is calculated cytopathy variability (such as the whole pathologies of each porocyte of a certain concentration, ratio is 1, and such as acellular pathology, then ratio is 0);
(4), calculate T=10 * 10
1+d (S-0.5)/ mL
D=Log
10Extent of dilution is (as being 10 times of extent of dilution, d=1)
Each concentration cytopathy ratio sum of S=.
2, result:
2.1, ZBED6 gene PCR amplification:
Take the Qinchuan Cattle genome as template, with reference to upper ox ZBED6 gene order (accession number: NC 007314.4) the design primer of announcing of NCBI.Utilize the PrimeSTAR archaeal dna polymerase, successfully amplify ZBED6, the result as shown in Figure 1, Fig. 1 is the Qinchuan Cattle ZBED6 gene of pcr amplification: wherein M is D15000Marker, and band is respectively 250,1000,2500,5000,7500,10000,15000bp; 1 is the ZBED6 gene fragment, and size is 2943bp.
2.2, pAdTrack-CMV-ZBED6 recombinant plasmid enzyme cuts qualification result:
Through connecting and expanding numerously, with KpnI and XbaI double digestion pAdTrack-CMV-ZBED6 recombinant plasmid, agarose detects the 2943bp fragment, prove and successfully obtain the positive recombinant plasmid of pAdTrack-CMV-ZBED6, the result as shown in Figure 2, Fig. 2 is that pAdTrack-CMV-ZBED6 recombinant plasmid double digestion is identified: M is D15000Marker, and band is respectively 250,1000,2500,5000,7500,10000,15000bp; 1 is KpnI and XbaI double digestion.
2.3, the structure result that carries the pAd-ZBED6 plasmid of ZBED6 gene:
As shown in Figure 3, carry the pAd-ZBED6 construction of recombinant plasmid success of ZBED6 gene.Cut evaluation by the PacI enzyme, further confirm to carry the pAd-ZBED6 construction of recombinant plasmid success of ZBED6 gene, the result as shown in Figure 3, Fig. 3 is that pAd-ZBED6 recombinant adenovirus plasmid PacI enzyme is cut evaluation: 1 cuts the small pieces degree of a 3.0kb of generation for the PacI enzyme; M is D15000Marker, and band is respectively 250,1000,2500,5000,7500,10000,15000bp.
2.4, the pAd-ZBED6 recombinant plasmid liposome LIPOFECTAMINE2000 transfection 293A cell effect of carrying the ZBED6 gene:
5d and 10d transfection effect are seen Fig. 4 and Fig. 5, and Fig. 4 is pAd-ZBED6 plasmid transfection 293A cell 5d, and Fig. 5 is pAd-ZBED6 plasmid transfection 293A cell 10d, prove the transfection success.
2.5, the pAd-ZBED6 recombinant plasmid liposome LIPOFECTAMINE 2000 transfection Qinchuan Cattles muscle cell cell effect of former generation of carrying the ZBED6 gene:
4d transfection effect is seen Fig. 6, and Fig. 6 is pAd-ZBED6 plasmid transfection Qinchuan Cattle muscle cell 4d of former generation, proves the transfection success.
2.6, carry the titer determination of the recombinant adenovirus of Qinchuan Cattle ZBED6 gene:
After the breeding of recombinant adenovirus poisons, the amplification, calculating virus titer is 1.96 * 10
10Pfu/mL illustrates that virus titer is higher, can satisfy transfectional cell and other application.
Embodiment 2: the Qinchuan Cattle muscle cell that carries the former culture of recombinant adenovirus transfection of Qinchuan Cattle ZBED6 gene:
The purpose of the present embodiment is recombinant adenovirus plasmid pAd-ZBED6 infected cattle muscle cell of former generation, crosses the differentiation of expression ZBED6 gene pairs muscle cell proliferation and the differential expression situation of muscle development marker gene (MyoD, MyoG, MEF-2D, Pax7, MHC) in the ox muscle cell through real-time quantitative PCR and Western bloting technology for detection.
1, the former culture of Qinchuan Cattle muscle cell:
(1), flesh tissue is placed culture dish, in aseptic operating platform, use Hank ' s liquid washing three times, and reject the foreign material such as fat, blood;
(2), with operating scissors with the muscle (1mm that is cut into small pieces
3), use again Hank ' s liquid to wash three times, be transferred in the little penicillin bottle;
(3), look 0.25% trypsin solution that the tissue block amount adds 5～6 times, digest 20～40min in 37 ℃, every the 5min vibration once, or with suction pipe piping and druming once, make cellular segregation;
(4), add 3～5mL nutrient solution to stop trysinization effect (or adding pancreatin inhibitor);
(5), leave standstill 5～10min, the tissue block of disperseing is sunk, get suspension and join in the centrifuge tube;
(6), 1000r/min, centrifugal 10min abandons supernatant liquor;
(7), add Hank ' s liquid 5mL, break up cell, once centrifugal again, abandon supernatant liquor;
(8), add nutrient solution l～2mL (visual cell's amount), blood counting chamber counting;
(9), cell is adjusted to 5 * 10
5About/mL, be transferred to 37 ℃ of cultivations in 75mL 2 square vases.
2, the RT-PCR behind the ZBED6 recombinant adenovirus transfection primary cell detects:
When the Qinchuan Cattle muscle cell density of former generation of cultivating reaches 60%, add an amount of viral supernatant cells infected.During cell infection virus (MOI=200) 72h, RT-PCR detects proof, compare (negative control is the recombinant adenovirus plasmid that empty pAdTrack-CMV and pAdEasy-1 restructuring produce, and does not carry the ZBED6 gene) with negative control, the expression amount of ZBED6 gene mRNA obviously increases.90% muscle cell is expressed green fluorescence simultaneously, and the muscle cell form is good.
Embodiment3:E.coli the preparation of DH5 α competent cell:
The preparation process of competent cell E.coli DH5 α in the embodiment of the invention:
1, step:
(1), common 4mLLB nutrient solution shakes bacterium and spends the night in the 15mL centrifuge tube;
(2), 1:100 inoculation next day enlarged culturing in 200mLRichLB (seeing Table 6) nutrient solution (37 ° of C) to OD
600=0.6～0.7;
(3), place 10min on ice, shake and make its cooling, minute install to 50mL centrifuge tube, every pipe 42mL;
(4), 4 ° of C, 2000r/min, 15min, collecting cell (removing most supernatant);
(5), the CCMB80Buffer (seeing Table 7) of 1/6 volume is resuspended, puts 20min on ice;
(6), 4 ° of C, 2000r/min, 15min, collecting cell (removing most supernatant);
(7), resuspended with 1/24 volume CCMB80Buffer, per 100 μ L are in a centrifuge tube ,-80 ° of C are frozen.
The component of table 6Rich LB
The prescription of table 7CCMB80Buffer
Among the CCMB80 all ingredients answer weighing accurate, use first a certain amount of ddH
2O dissolves each compound, adds glycerine again, stirs, and uses at last ddH
2The O constant volume is to volume required.CCMB80 can not autoclaving, pH is transferred to 6.4 after, use first filter paper filtering, then use 0.22 μ m membrane filtration, be stored in 4 ° of C.The above, it only is preferred embodiment of the present invention, be not that the present invention is done any formal and substantial restriction, all those skilled in the art, within not breaking away from the technical solution of the present invention scope, when can utilizing the disclosed above technology contents, and a little change of making, modify the equivalent variations with differentiation, be equivalent embodiment of the present invention; Simultaneously, the change of any equivalent variations that all foundations essence technology of the present invention is done above embodiment, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (10)
1. plasmid-type adenovirus carrier pAd-ZBED6, it is characterized in that: described plasmid-type adenovirus carrier pAd-ZBED6 contains the Qinchuan Cattle ZBED6 gene of nucleotide sequence shown in the SEQ ID NO.1.
2. plasmid-type adenovirus carrier pAd-ZBED6 as claimed in claim 1, it is characterized in that: described plasmid-type adenovirus carrier pAd-ZBED6 is finished by the AdEasyTM system constructing.
3. plasmid-type adenovirus carrier pAd-ZBED6 as claimed in claim 1, it is characterized in that: the ZBED6 gene among the described plasmid-type adenovirus carrier pAd-ZBED6 is connected under the shuttle plasmid CMV promotor.
4. plasmid-type adenovirus carrier pAd-ZBED6 as claimed in claim 1 is characterized in that: contain the Kozak sequence before the ZBED6 gene start codon among the described plasmid-type adenovirus carrier pAd-ZBED6.
5. plasmid-type adenovirus carrier pAd-ZBED6 as claimed in claim 1 is characterized in that: contain 6 His sequence labels behind the ZBED6 gene start codon among the described plasmid-type adenovirus carrier pAd-ZBED6.
6. method that makes up the described plasmid-type adenovirus carrier of arbitrary claim pAd-ZBED6 in the claim 1 to 6, described method comprises the steps:
(1), the ZBED6 gene fragment with pcr amplification is connected to the pGM-T carrier, recombinant plasmid pGM-T-ZBED6; With recombinant plasmid pGM-T-ZBED6 and shuttle plasmid pAdTrack-CMV through KpnI and XbaI double digestion, utilize T4DNALigase to carry out even reaction of enzyme ZBED6 gene fragment and pAdTrack-CMV carrier segments, enzyme connects product Transformed E .coli DH5 α competence bacterium, obtains the pAdTrackCMV-ZBED6 recombinant plasmid;
(2), restriction enzyme Pme I linearizing pAdTrack-CMV-ZBED6 recombinant shuttle plasmid, contain pAdEasy-1 plasmid E.coliBJ5183 competence bacterium with the conversion of linearizing pAdTrack-CMV-ZBED6 recombinant shuttle plasmid, utilize the efficient homologous recombination of Cre recombinase system in the E.coli BJ5183, in bacterium, finish the homologous recombination of pAdTrack-CMV-ZBED6 and pAdEasy-1, obtain to carry the recombinant adenovirus plasmid pAd-ZBED6 of ZBED6 gene.
7. method according to claim 6 is characterized in that: described method also comprise with recombinant adenovirus plasmid pAd-ZBED6 with liposome LIPOFECTAMINE 2000 transfections after, in the 293A cell, carry out virus and pack.
8. the described plasmid-type adenovirus carrier of arbitrary claim in the claim 1 to 5 is as the application of Qinchuan Cattle ZBED6 Functional identification of genes.
9. the described plasmid-type adenovirus carrier of arbitrary claim in the claim 1 to 5, the application of transforming as cell.
10. the described plasmid-type adenovirus carrier of arbitrary claim in the claim 1 to 5 is as the application of muscle growth developmental regulation.
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| CN103290122A (en) * | 2013-05-28 | 2013-09-11 | 西北农林科技大学 | Detecting method and kit of cattle ZBED6 (Zinc finger, BED-type containing 6) gene mononucleotide polymorphism |
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